Jesper Tholstrup Hansen:
Programmed -1 ribosomal frameshift is a translational recoding event used by many viruses to create a well defined stoichiometric relationship between translational products of its genome. This recoding event occurs at specific points during the process of translating its genome into its encoded proteins. It is believed that all features required for this frameshift process is location on the translated RNA. One of the requirements for programmed - 1 ribosomal frameshift is a downstream RNA structure, typically a pseudoknot, and it is possible that the helicase activity of the ribosome is responsible for “untying” these structures following the frameshift event. Single molecule experiments have demonstrated that, at least for similar structures, a correlation exists between the mechanical strength of these pseudoknots and their ability to induce -1 frameshift during translation.
This thesis have focused on describing functional and physical aspects of completely artificial RNA pseudoknots and have demonstrated that structures with sufficiently high stability is able to stall translating ribosomes on the messenger RNA. This phenomenon had previously been suggested based on single molecule experiments, but is shown here for the first time.
Single molecule mechanical unfolding of RNA structures using force spectroscopy demonstrated that most of the artificial pseudoknots were able for fold as expected and our results were not in disagreement with the correlation between mechanical strength and ability to induce -1 frameshift. Additionally, our sinle molecule experiments showed that the folding dynamics of RNA is complex. A simulation tool was constructed to aid in the quantification of possible sources of error and to help classify the observed unfolding events.
Attempts were made to disrupt the function of the putative ribosomal helicase by creating point mutations in ribosomal proteins S3, S4 and S5 in Escherichia coli. Although no effect was observed on -1 frameshift, the putative helicase is still an interesting target, which deserves future attention.